1. Field of the Invention
The present invention relates generally to the field of protein engineering. More particularly, it concerns compositions comprising Fc antibody domains conferring increased binding to C1q relative to wild-type Fc antibody domains.
2. Description of Related Art
Currently, the top 25 marketed recombinant therapeutic antibodies have sales of well over $43.5 billion/year, and with a forecasted annual growth rate of 9.2% from 2010 to 2015, they are projected to increase to $62.7 billion/year by 2015 (Elvin et al., 2013). Monoclonal antibodies (mAbs) comprise the majority of recombinant proteins currently in the clinic, with 1064 products undergoing company-sponsored clinical trials in the USA or EU, of which 164 are phase III (Elvin et al., 2013). In terms of therapeutic focus, the mAb market is heavily focused on oncology, arthritis, and immune and inflammatory disorders, and products within these therapeutic areas are set to continue to be the key growth drivers over the forecast period. As a group, genetically engineered mAbs generally have a higher probability of FDA approval success than small-molecule drugs. At least 50 biotechnology companies and all major pharmaceutical companies have active antibody discovery programs in place. The original method for isolation and production of mAbs was first reported at 1975 by Milstein and Kohler (Kohler and Milstein, 1975), and it involved the fusion of mouse lymphocyte and myeloma cells, yielding mouse hybridomas. Therapeutic murine mAbs entered clinical study in the early 1980s; however, problems with lack of efficacy and rapid clearance due to patients' production of human anti-mouse antibodies (HAMA) became apparent. These issues, as well as the time and cost consumption related to the technology, became driving forces for the evolution of mAb production technology. Polymerase Chain Reaction (PCR) facilitated the cloning of monoclonal antibody genes directly from lymphocytes of immunized animals and the expression of combinatorial libraries of antibody fragments in bacteria (Orlandi et al., 1989). Later libraries were created entirely by in vitro cloning techniques using naive genes with rearranged complementarity determining region 3 (CDR3) (Griffths and Duncan, 1998; Hoogenboom et al., 1998). As a result, the isolation of antibody fragments with the desired specificity was no longer dependent on the immunogenicity of the corresponding antigen. Moreover, the range of antigen specificities in synthetic combinatorial libraries was greater than that found in a panel of hybridomas generated from an immunized mouse. These advantages have facilitated the development of antibody fragments to a number of unique antigens including small molecular compounds (haptens) (Hoogenboom and Winter, 1992), molecular complexes (Chames et al., 2000), unstable compounds (Kjaer et al., 1998), and cell surface proteins (Desai et al., 1998). In microbial cells, display screening may be carried out by flow cytometry. In particular, Anchored Periplasmic Expression (APEx) is based on anchoring the antibody fragment on the periplasmic face of the inner membrane of E. coli followed by disruption of the outer membrane, incubation with fluorescently-labeled target, and sorting of the spheroplasts (U.S. Pat. No. 7,094,571). APEx was used for the affinity maturation of antibody fragments (Harvey et al., 2004; Harvey et al., 2006). In one study, over 200-fold affinity improvement was obtained after only two rounds of screening.
One important mechanism underlying the potency of antibody therapeutics is Fc-mediated effector functions for clearance of a target antigen (or cell) via two processes. The Fc domain binds to a number of proteins including soluble proteins in serum and receptors on cell surfaces. Binding of the Fc region of antibodies that have formed immune complexes with a pathogenic target cell to the complement protein C1q, result in the activation of the classical complement activation cascade (Walport, 2001; Janeway et al., 2005). Separately, the Fc domain binds to different receptors expressed on the surface of leukocytes to elicit antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP).
In particular, activation of the classical pathway following the formation of a complex between C1q and antibodies bound to pathogen elicits a cascade of biochemical reaction that lead to pathogen elimination via several mechanisms. First, formation of the membrane attack complex (MAC) on the surface of the cell, which kills cells by comprising the integrity of the cell membrane. Second, opsonization due to the deposition of complement proteins onto the surface of the pathogen and recognition of the complement opsonins by complement receptors on leukocytes triggers complement dependent cell cytotoxicity (CDCC). A single molecule of IgG can not activate the complement pathway because of the low affinity of IgG for C1q and because the requirement that C1q binds to multiple IgG molecules in the proper spatial orientation (i.e., as an immune complex) in order to initiate conformational changes necessary for the activation of the so called “classical” complement pathway (Walport, 2001; Janeway et al., 2005).
In humans there are two general classes of FcγRs that bind to the Fc domain of IgG subclass antibodies: activating receptors, characterized by the presence of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) sequence, and the inhibitory receptor, characterized by the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence (Daeron, 1997; Bolland et al., 1999). Of note, activating FcγRs (i.e., FcγRI, FcγRIIA, FcγRIIIA, and FcγRIIIB) induce activating or pro-inflammatory responses, while the inhibitory receptor (i.e., FcγRIIB) induces anti-inflammatory responses. The ability of antibodies to induce activating ADCC depends on the ratio of binding affinities to the activating FcγRs vs. the inhibitory FcγRIIB (A/I ratio) (Boruchov et al. 2005; Kalergis et al., 2002). A number of allotypes of the FcγRs are known. For example, the FcγRIIAH131 allotype shows higher binding affinity for IgG than the FcγRIIAR131 allotype, while the FcγRIIIAV158 allotype shows higher binding affinity than FcγRIIIAF158IgG1 Fc domains, which bind to both the activating and the inhibitory FcγRs as well as to C1q. In contrast, human IgG2 isotype antibodies bind weakly to C1q (and thus are very poor in mediating complement activation and show little or no binding to FcγRs). Human IgG3 and IgG4 isotype antibodies display respectively higher and no C1q binding relative to IgG1 and generally weaker affinity to FcγRs.
The C1q and FcγR binding sites on IgG1 have been identified based on, docking models of IgG1 with C1q and crystal structures of the Fc domain with the extracellular domains of the FcγRs. Both C1q and FcγRs interact primarily with amino acids located in the Fc CH2 domain and in some instances the hinge of IgG1 antibodies. For C1q, Asp270, Lys322, and Pro329-Pro331 are particularly important for binding, as is the orientation of the Fab arms (Gaboriaud et al., 2003; Guddat et al., 1993) In terms of FcγR binding, Leu234-Ser239 in the IgG lower hinge region and Asp265-Ser267 in the CH2 domain are particularly important (Gaboriaud et al., 2003; Woof et al., 2004). The CH2 domain has one N-glycosylation site at Apn297, and the N-linked glycan at Asn297 bridges the gap between the two CH2 domains. This bridge maintains the proper conformation of CH2 domains for binding to C1q and FcγRs. On the other hand, the removal of the glycan at Asn297 increases the conformational flexibility of the CH2 domains, and as a result, aglycosylated Fc show essentially no binding to C1q and to FcγRs, thus abolishing ADCC and CDC (Borrok et al., 2012).
Antibody mediated complement activation and CDC are of particular importance in the function of numerous therapeutic antibodies (Rogers et al., 2014). Therefore strategies to increase complement activation have received considerable attention. For example, chimeric IgG molecules comprising IgG1 and IgG3 (Natsume et al., 2008) were reported to display enhanced C1q binding without affecting FcγRs-binding ability resulting in enhanced CDC activity towards CD20+ lymphoma cell lines. Dall'Acqua et al. (2006) reported that amino acid substitution in the hinge region of human IgG1 resulted in slightly decreased CDC activity and lower ADCC activities compared to wild-type IgG. In another study, a K326W/E333S double mutation in the IgG1 Fc domain resulted in a 5-fold increase in binding to C1q and 2-fold increase in CDC (Idusogie et al., 2001). More recently Moore et al. (2010) reported that a S267E/H268F/S324T triple mutant in the IgG1 Fc domain showed 47-fold-enhanced affinity to C1q and 6.9-fold-enhanced EC50 values in CDC. The S267E/H268F/S324T triple mutant also showed increased affinity towards some but not all of the human FcγRs (Moore et al., 2010). Finally, Diebolder et al. (2014) reported that mutations in the Fc domain that favor the formation of IgG hexamers, most notably an E345R substitution, showed 12-fold enhanced CDC efficacy towards CD20+ positive Daudi cells. However, the effects of hexamer formation on FcγR binding or ADCC has not been reported.
E. coli possesses a reducing cytoplasm that is unsuitable for the folding of proteins with disulfide bonds, which accumulate in an unfolded or incorrectly folded state (Baneyx and Mujacic, 2004). In contrast to the cytoplasm, the periplasm of E. coli is maintained in an oxidized state that allows the formation of protein disulfide bonds. Notably, periplasmic expression has been employed successfully for the expression of antibody fragments, such as Fvs, scFvs, Fabs, or F(ab′)2s (Kipriyanov and Little, 1999). These fragments can be made relatively quickly in large quantities with the retention of antigen binding activity. However, because antibody fragments lack the Fc domain, they do not bind the FcRn receptor and are cleared quickly; thus, they are only occasionally suitable as therapeutic proteins (Knight et al., 1995). Until recently, full-length antibodies could only be expressed in E. coli as insoluble aggregates and then refolded in vitro (Boss et al., 1984; Cabilly et al., 1984). Clearly this approach is not amenable to the high-throughput screening of antibody libraries since with the current technology it is not possible to refold millions or tens of millions of antibodies individually. A further problem is that since E. coli expressed antibodies are not glycosylated, they fail to bind to complement factor 1q (C1q) or Fc and other Fcγ receptors with the exception of the neonatal Fc receptor (FcRn), which is critical for the long persistence of IgG antibodies in circulation.